KR20060035432A - Marker proteins for the diagnosis of exposure to dioxin and the diagnosis method using the same - Google Patents

Abstract

The present invention is a marker protein for diagnosing the exposure of tetrachlorodibenzodioxin (2,3,7,8-tetrachlorodibenzo- p -dioxin, hereinafter abbreviated as 'TCDD') and a dioxin exposure using the same. In particular, the present invention relates to a method for identifying and identifying marker proteins in which a sugar chain increases or decreases upon dioxin exposure, and confirms dioxin exposure using a sugar chain change of the marker protein. Marker protein for diagnosing dioxin exposure of the present invention and a method for confirming dioxin exposure using the same can be usefully used for diagnosing dioxin toxicity.

Dioxin, glycomix, sugar chain change, TCDD

Description

Marker proteins for the diagnosis of exposure to dioxin and the diagnosis method using the same}             

1 is a schematic diagram showing a lectin that specifically binds to each of the N-linked sugar chains,

FIG. 2 is a schematic diagram illustrating a series of experiments performed by performing mass spectrometry after performing LCA (Lens culinaris agglutinin) lectin affinity chromatography and performing two-dimensional electrophoresis.

Figure 3 shows the expression of sugar chains in Chang cell line treated with DMSO treated control and tetrachlorodibenzodioxin (2,3,7,8-tetrachlorodibenzo- p -dioxin, hereinafter abbreviated as 'TCDD'). These are two-dimensional electrophoresis (pH 3-10 strips) photographs taken to find increasing or decreasing proteins,

FIG. 4 is a two-dimensional electrophoresis (pH 4-7 strip) photograph performed to find a protein having increased or decreased expression of sugar chains in a DMSO-treated control group and a TCDD-treated Chang cell line.

The present invention is a marker protein for diagnosing the exposure of tetrachlorodibenzodioxin (2,3,7,8-tetrachlorodibenzo- p -dioxin, hereinafter abbreviated as 'TCDD') and a dioxin exposure using the same. In more detail, the present invention relates to a dioxin-exposed diagnostic marker protein and a dioxin-exposed method, which are identified by identifying proteins that increase or decrease sugar chains when TCDD is applied to human hepatocyte cell Chang cells. It is about how to.

Representative environmental hormones include dioxins, PCBs, DDT, organochlorine pesticides, heavy metals, and plastic plasticizers. Among them, dioxins are considered to be the most toxic and dangerous environmental pollutants or toxic compounds. Average dioxin (dioxin) as if, polyclonal rineyi suited dibenzo p-dioxin (polychlorinaged dibenzo- p -dioxins, hereinafter abbreviated to 'PCDDs') and polyclonal rineyi suited dibenzofuran acids (polychlorinated dibenzofuran, hereinafter 'PCDFs' Family is known to date, so far about 75 species of PCDD and about 135 species of isomers have been identified.

Dioxins are stable substances that are not chemically decomposed and are easily adsorbed on the surface of particulate matter such as dust, ash, soil, etc., and once combined, they do not easily separate, which increases environmental pollution and accumulates in animals and plants. Dioxins come from industrial manufacturing processes such as plating and steelmaking, chemical manufacturing processes such as pesticides, incineration of household waste and industrial wastes, and chlorine bleaching processes of paper and pulp mills. Dilute or soak into soil, plants, or water. Dioxin exposure pathways in adults account for over 95% of all food intake, including meat, fish, milk and fat, and are also exposed through breathing and contact with soil. Breastfeeding children are also exposed to dioxin through breastfeeding.

Dioxin molecules introduced into the body bind to specific receptor sites (Aryl Hydrocarbon Receptor, AhR) in the cell tissue, acting like hormones to the cells, or hormones or regulators that normally bind due to dioxin do not bind to the receptors. As a result, cellular functions do not work properly. Accumulation of dioxin in the human body leads to reproductive function (uterine bleeding and pain, infertility, sperm reduction) and immune function abnormalities. ), Peripheral and central nervous system developmental disorders are known to cause.

Although studies on toxic substances including dioxin have been actively conducted, recently, high-resolution gas chromatography / mass spectrometry (HRGC / HRMS) was the only dioxin detection method (C. Rappe et al. Envrion Sci Technol. 21: 964, 1987). However, with the rapid development of biotechnology over the last decade, in vitro assays and ligand binding assays have been developed, and EROD-bioassay, AHH bioassay, Enzyme immunoassay (EIA), CALUX assay, Various methods have been developed, including the GRAB assay (PA Bechisch et al. Environment International. 27: 413, 2001). Most of these methods use a CYP450 1A1 protein called mixed-function oxidase. CYP450 1A1 is the first protein to be exposed to dioxin and is suitable for dioxin exposure and toxicity testing. However, the expression of other toxic substances other than dioxins have been reported, which is not specific to dioxins.

Meanwhile, various methods for analyzing the function of proteins have been tried. Recently, as mass spectrometers such as Matrix-Assisted Laser Desorption Ionization (MALDI-TOF) have been developed, proteomics has come into the spotlight as a method of protein function analysis. have. However, proteomics is not suitable for analyzing post-translational modifications of complex signal transduction because it selectively analyzes only one stationary time point for a dynamically moving organism. In order to solve this problem, there is a method using lectin, which refers to a carbohydrate-binding protein that specifically binds to a monosaccharide or oligosaccharide, and has a tendency to bind to a glycolipid or glycoprotein sugar chain in vivo. Glycomics, which combines the analysis of lectins with sugar chain binding to the proteomics, is called glycomics, which tracks the glycosylation of proteins after translation or during modification, and takes it one step further by overcoming proteomic difficulties. Is considered.

Examples of disease diagnosis using glycosylation of proteins include the case of α-fetoprotein. N -acetyl-β-glucosaminide α1,6-fucosyltransferase ( N- acetyl-β-glucosaminide α1,6-fucosyltransferase, abbreviated α1,6-FucT '), an enzyme that catalyzes the attachment of GDP-fucose to the N-terminal glycoprotein. Α1,6 fucosylation occurs in α-fetoprotein by α1,6-FucT, resulting in α1,6-fucosylated α-fetoprotein (1,6-fucosylated). α-fetoprotein, hereinafter abbreviated as 'AFP', is produced and used to diagnose early liver cancer by observing the sugar chain changes of AFP (Naoyuki Taniguchi et al . Biochimica et Biophysica Acta, 1473: 9-20, 1998).

In the present invention, it was confirmed that there is a protein to which α1,6 fucose sugar chain branches are added or removed even in hepatocytes exposed to TCDD such as α-fetoprotein, and a method of using the same as a dioxin detection marker was studied. First, the glycoproteins that cause the sugar chain change upon dioxin exposure were identified by using glycomixes that have been taken one step from the existing proteomics method. Since the protein identified using the above method is on the surface of the cell or secreted form, it is possible to diagnose TCDD exposure through the diagnosis of body fluids such as blood or urine.

The present inventors completed the present invention by confirming that TCDD was treated to Chang cell line to find a protein having an increase and a decrease in sugar chain change, and to determine whether TCDD was exposed by measuring the sugar chain change of the protein. .

It is an object of the present invention to provide a method for identifying proteins that cause sugar chain changes in hepatocytes when exposed to dioxin and identifying dioxin exposure using the same.

In order to achieve the above object, the present invention provides a marker protein for diagnosing whether or not tetrachlorodibenzodioxin (2,3,7,8-tetrachlorodibenzo- p -dioxin, hereinafter abbreviated as 'TCDD').

In addition, the present invention provides a method for determining the exposure to TCDD by measuring the sugar chain change of the marker protein.

Hereinafter, the present invention will be described in detail.

The present invention whether or not (hereinafter referred to 2,3,7,8-tetrachloro dibenzo- p -dioxin, hereinafter 'TCDD') tetrachloro-dibenzo dioxin exposure provides a diagnostic marker protein.

In a preferred embodiment of the present invention, in order to find a protein that can be used as a dioxin marker, a protein identification experiment was performed by selecting liver cells, which are places where toxic substances accumulate in body organs.

Specifically, serum-free media of the experimental group treated with TCDD and DMSO-treated control cells were prepared in Chang cell lines, which are human liver cells. By loading this in a column packed with LCA (Lens Culinaris agglutinin), a type of lectin that binds to the α1,6-fucose sugar chain branch, changes in the α1,6-fucose sugar chain branch due to TCDD exposure The proteins that occurred were selected first. After the protein sample was eluted, one-dimensional electrophoresis (IEF) was performed by loading on a dry strip and two-dimensional electrophoresis (SDS-PAGE) was performed on the strip after the one-dimensional electrophoresis. Gels obtained by electrophoresis were subjected to staining and scanning to analyze the difference in protein expression using PDQuestTM, a gel analysis program.

The inventors have found a spot indicating the difference in expression from an image analysis program and the mass of the corresponding protein is known by a mass spectrometer (Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry, MALDI-TOF-TOF MS). Came out. Based on this, the peptide was identified in the NCBI database (see FIG. 2 ).

As a result, the proteins showing an increase or decrease in sugar chain change when TCDD was treated were as follows (see FIGS . 3 and 4 ).

First of all, a protein having an increased alpha 1,6-fucose sugar chain branch, an aldehyde dehydrogenase having a molecular weight of about 50 kDa and a pi of about 6.1, of about 38 kDa 'Cathepsin B' having a molecular weight and a pi of about 5.9, 'Collagen alpha I chain precursor' having a molecular weight of about 25 kDa and a pi of about 5.9, a molecular weight of about 16 kDa and about Lysozyme A with a pI of 9.4 and FK-506 binding protein 4 with a molecular weight of about 52 kDa and a pi of about 5.35 were identified.

Next, a protein having a reduced alpha 1,6-fucose sugar chain branch is a 'Protien disulfide isomerase' having a molecular weight of about 57 kDa and a pi of about 4.8. A 'lysozyme C precursor' having a molecular weight of 16 kDa and a pi of about 9.4, an 'Immunoglobulin heavy chain protein' having a molecular weight of about 14 kDa and a pi of about 5.0 was identified.

Since the glycoproteins exhibiting the sugar chain changes revealed above are secreted to the surface of the cells or secreted out of the cells, it is possible to easily diagnose the TCDD exposure by checking the sugar chain changes of the protein even with body fluids such as blood or urine.

In addition, the present invention provides a method for determining the exposure to TCDD by measuring the sugar chain change of the marker protein. That is, the change in sugar chain branch of a protein showing a change in α1,6 fucose sugar chain branch in cells exposed to dioxin compared to normal cells.

In general, liver cancer is diagnosed as an early liver cancer by a sugar chain change in which α1,6 fucose is bound to α-fetoprotein, and in the present invention, α1, By identifying proteins with 6 fucose sugar chain branches, it was confirmed that exposure to TCDD can be determined by only changing the sugar chains of the protein.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Sample Preparation

The present inventors prepared samples for electrophoresis by treating tetrahedral dibenzodioxin (2,3,7,8-tetrachlorodibenzo- p -dioxin, hereinafter abbreviated as 'TCDD') to human hepatocytes.

Specifically, 1000 ml of serum-free media of the experimental group treated with 50 nM TCDD for 72 hours and the control group treated with DMSO for 72 hours were collected on a Chang cell line, which is a human hepatocyte, and the LCA (Lens) was collected. A column (Sigma, # L-0511) packed with Culinaris agglutinin resin was loaded. 0.4 M methyl-alpha-D-manopyranoside and 50 mM Tris-Cl were added to the column to elute the glycoproteins attached to the resin. Proteins below 10 kDa were removed from the glycoprotein eluted using Ultrafree-4 centrifugal filter device (Millipore), concentrated to 2 ml, 20% TCA dissolved in acetone was added and precipitated at -20 ° C for 1 hour. The protein was recovered. This secured a protein sample that caused the sugar chain change after TCDD treatment ( FIG. 2 ).

Example 2 Two-Dimensional Electrophoresis

<2-1> One-Dimensional Electrophoresis

The present inventors performed one-dimensional electrophoresis (IEF) with the sample prepared in <Example 1> . Specifically, the protein precipitated in Example 1 was dissolved in a rehydration buffer (7 M Urea, 2M Thiourea, 4% CHAPS, 1% DTT, 0.5% IPG buffer) for 2 hours, and then dissolved at 12000 rpm. The supernatant was collected by centrifugation for 15 minutes. After hydration of the drystrip for 16 hours, the supernatant collected after centrifugation was loaded for 1 minute at 300V, 1 hour 30 minutes at 3500V, and 13 hours at 3500V Multiphor II (Pharmacia Biotech). One-dimensional electrophoresis was performed with.

<2-1> two-dimensional electrophoresis

The inventors performed two-dimensional electrophoresis (SDS-PAGE) with a strip of one-dimensional electrophoresis. Specifically, the strip prepared in Example <2-1> 5 ml of an equilibration solution (6M Urea, 1.5M Tris-Cl (pH 8.8), 30% Glycerol, 2% SDS, 5% β-Mercaptoethanol, Bromophenol blue slightly) After reacting for 15 minutes, it was placed on 11% polyacrylamide gel (14 × 15 cm). Electrophoresis buffer (24 mM Tris, 250 mM glycine, 0.1% SDS) was added to HoferTM SE600 (Pharmacia Biotech) and subjected to two-dimensional electrophoresis for 6 hours at 15 mA 1 hour and 30 mA 5 hours per sheet. ( FIG. 2 ).

<2-3> Dyeing and Image Analysis

We stained gels subjected to two-dimensional electrophoresis and analyzed the spots on the gel with a gel analysis program.

Specifically, the gel was separated from the glass plate, washed three times with 15 minutes of distilled water, and reacted for 12 hours while adding a dye Bio-SafeTM coomassie (Bio-Rad). After completion of the reaction, the gel was again washed with distilled water and scanned (scanning), the protein difference was analyzed by PDQuestTM (Bio-rad), a two-dimensional electrophoretic gel analysis program ( Fig. 3 and 4 ).

Example 3 Mass Analysis

<3-1> Sample Preparation for Mass Spectrometry

We identified proteins that correspond to spots obtained from image analysis programs.

Specifically, the spot was cut from the gel after two-dimensional electrophoresis, treated with 30% methanol, and 100 μl of a solution containing 10 mM ammonium bicarbonate in 50% acetonitrile. Was treated again to completely remove the dye. After the gel was dehydrated with 100 μl of 100% acetonitrile, the remaining solution was completely removed in a vacuum centrifuge (Speed-vac). 10 ng / μl of trypsin was treated to the dehydrated gel and reacted at 37 ° C. for 16 hours to cut proteins in the gel into peptides. 100 μl of 50 mM ammonium bicarbonate was added thereto and reacted at 37 ° C. for 1 hour, and then the peptide solution was collected. Then, 100 μl of 50% acetonitrile and 5% trifluoroacetic acid (TFA) were added to the gel. After reacting for 1 hour at 37 ° C., the peptide solution was collected to extract the peptide from the gel. The extracted peptide was completely dried in a vacuum centrifuge.

Zip-Tip (Millipore) washed three times with 10 μl with 100% ACN, three times with 10 μl of 50% ACN and 0.1% TFA, and three times with 0.1% TFA to remove salt from the sample prior to mass spectrometry. G) was bound to a sample dissolved in 10 µl of distilled water. This was washed three times with 10 μl of 0.1% TFA three times, and then 50 μl ACN and 10 μl of 0.1% TFA were added again to elute the peptide from the Zip-Tip, thereby completely removing the salt from the peptide. This prepared a peptide sample for mass spectrometry.

<3-2> mass spectrometry

The inventors put the sample prepared in Example <3-1> into a mass spectrometer (Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry, MALDI-TOF-TOF MS) and performed mass spectrometry.

Specifically, alpha-cyano-4-hydroxycinnamic acid (CHCA) used as a matrix is dissolved in 50% ACN and 0.1% TFA, followed by centrifugation, and the supernatant. Only collected. The matrix mainly uses organic material that absorbs ultraviolet rays strongly, and serves to separate each sample molecule and to stably soft ionize it. Dilute the standard solution for internal calibration to 1/4000 in the matrix, add 1 μl of the solution to the sample, dissolve it, and drop the sample onto the analytical plate. Was carried out. The mass spectrometer used a Voyager DE-STR Mass spectrometer (Applied Biosystems). The mass of the peptide obtained by mass spectrometry was analyzed in Mascot ( http://www.matrixscience.com ), and the peptide was identified in the NCBI database.

As a result, proteins with increasing or decreasing sugar chain changes were identified as shown in FIGS . 3 and 4 . Proteins exhibiting sugar chain changes are as follows.

1. Protein to which alpha, 1,6-fucose sugar chain branch is added

point Molecular Weight (kDa / pI)   protein 4 57 / 4.8   Protein disulfide isomerase 5,6 16 / 9.4   Lysozyme C precursor 9 14 / 5.0   Immunoglobulin heavy chain

2. Proteins with reduced alpha, 1,6-fucose sugar chains

point Molecular Weight (kDa / pI)   protein One 50 / 6.1   Aldehyde dehydrogenase 2,3 38 / 5.9   Cathepsin b 7 25 / 5.9   Collagen alpha 1 (Ⅰ) chain precursor 8 16 / 9.4   Lysozyme A 10 52 / 5.35   FK-506 binding protein 4

The sugar chain change of the protein identified above can be measured to diagnose the exposure of TCDD.

As described above, a method for diagnosing whether or not tetrachlorodibenzodioxine (2,3,7,8-tetrachlorodibenzo- p -dioxin, TCDD) is exposed using a sugar chain change of the protein and a marker protein for diagnosing the exposure are as follows. In addition to being used to diagnose dioxin exposure, monoclonal antibodies prepared based on the marker protein may be useful for developing protein chips and diagnostic kits using immune responses.

Claims (4)

Tetrachlorodibenzodioxin (2,3,7,8-tetrachlorodibenzo- p -dioxin, hereinafter abbreviated as 'TCDD'). The method of claim 1, wherein the marker protein is 'aldehyde dehydrogenase', 'Cathepsin B', 'Collagen alpha I chain precursor', 'Lysozyme A' ',' FK-506 binding protein 4 ',' Protien disulfide isomerase ',' Lysozyme C precursor 'and' Immunoglobulin heavy chain protein ' And a protein selected from. A method for determining the exposure to tetrachlorodibenzodioxin (2,3,7,8-tetrachlorodibenzo- p -dioxin, TCDD) by measuring the sugar chain change of the marker protein of claim 1. 4. The method of claim 3, wherein the sugar chain change is a change in which α1,6-fucose sugar chain branches are added or reduced.

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